Polyenoic acid metabolism in cultured human skin fibroblasts

The incorporation of [1-¹⁴C]linoleic acid, and [1-¹⁴C]linoleic acid into cellular lipids of cultured human skin fibroblasts was studied. Cultured cells took up both labeled fatty acids at nearly the same rate and incorporated them into a variety of lipid classes. At the end of 1 hr incubation with [1-¹⁴C]linoleic acid, radioactivity was found in the triacylglycerol (TG) and choline phosphoglyceride (CPG) pools preferentially. Incorporation into the TG fraction decreased rapidly, while the uptake into CPG, serine phosphoglyceride (SPG), and ethanolamine phosphoglyceride (EPG) fractions increased progressively with longer incubation times. Similar results were obtained with [1-¹⁴C]linoleic acid as precursor. At the end of 24 hr, desaturation and chain elongation of 18∶3 n−3 was more extensive than conversion of 18∶2 n−6 to higher polyenoic acids. During pulse-chase experiments with either fatty acid precursor, the incorporated radioactivity was progressively lost from cellular lipids, particularly from the TG and CPG fractions, but continued to increase in the SPG and EPG pools. The similar labeling pattern of cellular phospholipids with linoleic or linolenic acids, and data from pulse-chase studies suggest that a direct transfer of fatty acids from CPG to EPG is a likely pathway in fibroblast cultures. Incorporation into the EPG pool during the pulse-chase experiments paralleled extensive desaturation and elongation of linoleic acid into 20∶4 n−6, and 22∶4 n−6; and of linolenic acid into 22∶5 n−3 and 22∶6 n−3.     

Acetate and mevalonate labeling studies with developingCuphea lutea seeds

Cuphea seeds contain large amounts of medium chain (C₈ to C₁₄) fatty acids, mainly as triacylglycerols. The biosynthesis of these lipids was studied in vivo by incubating developingCuphea lutea seeds with labeled acetate. Incorporation of label into triacylglycerols and into medium chain fatty acids occurred principally during the period of endogenous lipid deposition, but some label was encountered in these products even during seed dehydration. At this later stage palmitate and oleate were the dominant labeled fatty acids. During the period of rapid endogenous lipid deposition acyl lipids other than triacylglycerols were minor labeled components. The labeling patterns were consistent with the Kennedy pathway for triacylglycerol biosynthesis. The fatty acid composition of the acyl-CoA pool was similar to the total lipid fatty acid composition, but the acyl-ACP pool contained relatively more short chain acyl groups. Squalene was labeled from acetate throughout the period of seed development, but labeled sterols were not detected. Using [2-¹⁴C]mevalonic acid lactone as substrate, squalene was the principal labeled product. Small amounts of label were found in free sterols. However, in terms of mass, free sterol dominated over squalene. The possibility of two independent sites of isoprenoid biosynthesis in the developing embryo is discussed.     

Fatty acid synthesis in the oil palm (Elaeis guineensis): Incorporation of acetate by tissue slices of the developing fruit

Oil palm (E. guineensis) fruits at three stages of development were studied. At week 12–13 after anthesis, the endosperm had started accumulating oil and tissue slices incorporated [1-¹⁴C] acetate into fatty acids which resembled those found in the mature endosperm. The mesocarp contained very little oil and incorporated acetate into polar lipids. At week 16–17, the mesocarp started to accumulate oil; this was reflected in the [¹⁴C] lipid products from acetate incubation. At or just prior to this stage, an increase in the endogenous linoleic and linolenic acid content and the increase in fruit size indicated cellular growth in the mesocarp tissue. At week 20–21 the fruit was ripe, and both endosperm and mesocarp tissues were filled with storage oil. [¹⁴C] Fatty acids synthesized from acetate by mesocarp slices at this stage were the same as the endogenous storage fatty acids in bothE. guineensis andE. oleifera. A very weak fatty acid synthesizing activity was seen in the mature endosperm, but the products had no relationship to the storage lipid.     

Tissue culture of cocoa beans (Theobroma cacao L.): Incorporation of acetate and laurate into lipids of cultured cells

Suspension cultures of cocoa bean tissue readily incorporated exogenous acetate into lipids. The distribution of radioactivity from acetate in individual lipid classes after 48 hr was 20, 5, 1, 15, 25, and 35% in triglycerides, diglycerides, free fatty acids, sterol esters, sterols and polar lipids, respectively. The labeled acetate was rapidly incorporated into various fatty acids within 2 hr. The [1-¹⁴C] saturated fatty acids declined slightly after 4 hr, whereas [1-¹⁴C] oleate declined significantly after 2 hr. There was a concomitant increase in [1-¹⁴C] linoleate. The radioactivity associated with linolenate was relatively high up to 4 hr, declined by 24 hr, and then increased again. The kinetics of fatty acid labeling suggested that biosynthesis of linolenic acid in cocoa bean suspension culture may occur via the desaturation of linoleic acid and the chain elongation of dodecatrienoic acid. The patterns of fatty acid radiolabeling following incubation of cells with [1-¹⁴C] laurate was consistent with this mechanism.     

Varietal difference in lipid content and fatty acid composition of highbush blueberries

Lipids from five cultivars of highbush blueberries (Vaccinium corymbosum L.) were extracted and fractionated into neutral lipids (60–66%), glycolipids (20–22%) and phospholipids (14–18%). The major fatty acids in all fractions were palmitic (16∶0), oleic (18∶1), linoleic (18∶2), and linolenic (18∶3) acids. All lipid classes had a large concentration of C₁₈ polyunsaturated acids (84–92%), indicating that blueberries are a rich source of linoleic and linolenic acids. Changes in the fatty acid composition of neutral lipids and phospholipids were not significantly different among the five cultivars, but significant differences were noted in the ratios of linoleic and linolenic acids in the glycolipids fraction.     

Preferential oxidation of linolenic acid compared to linoleic acid in the liver of catfish (Heteropneustes fossilis andClarias batrachus)

The fate of [1-¹⁴C] linoleic acid and [1-¹⁴C] linolenic acid in the liver slices and also in the liver tissues of live carnivorous catfish,Heteropneustes fossilis andClarias batrachus, was studied. Incorporation of the fatty acids into different lipid classes in the live fish differed greatly from the tissue slices, indicating certain physiological control operative in vivo. The extent of desaturation and chain elongation of linoleic and linolenic acids into long-chain polyunsaturated fatty acids was low. Linolenic acid was oxidized (thus labeling the saturated fatty acid with liberated¹⁴C-acetyl-CoA) in preference to linoleic acid, and this oxidation also seemed to be under physiological control since both of the fatty acids were poorly oxidized in the tissue slices and in the killed fish. These fish can therefore recognize the difference in the acyl chain structures of linoleate and linolenate. The higher oxidation of liolenic acid and poor capacity for its conversion to longer chain, highly unsaturated derivatives indicates a higher demand for the dietary supply of these essential fatty acids in these two species.     

The behavior of rat bile phospholipids in the intestine and in incubation media containing pancreatic juice

Samples of radioactive bile were collected from rats after intravenous injection of potassium soaps ([9–10³H₂] or [1¹⁴C] oleate, [1¹⁴C] linoleate or [9–10³H₂] palmitate). These radioactive acids were chosen because it is well established that, in natural phosphatidyl cholines, palmitic acid is located chiefly at the 1 position and linoleic and oleic acids at the 2 position. After incubation of bile with pancreatic juice, the labeling of unchanged biliary phospholipids was higher when native bile was labeled with oleic acid than with palmitic or linoleic acids. These data suggest that monounsaturated molecular species of biliary phospholipids are more resistant than the diunsaturated ones to in vitro hydrolysis by phospholipase A₂. Ninety min after introduction of the radioactive bile into the upper part of the rat duodenum, high labeling of luminal phospholipids was observed regardless of the bile sample used, although labeling of free fatty acids was always low. The passage of intact biliary phospholipids through the intestinal epithelium is discussed.     

Canavalia ensiformis neutral lipids,a rich source of lupeol

The composition of the neutral lipids ofCanavalia ensiformis, which represent 2.21% of whole seeds, has been investigated. The fatty acid composition is characterized by the presence of palmitic (15%), oleic (54%), linoleic (7%) and linolenic (8%) acids. The unsaponifiable matter (7.9% of the neutral lipids), was examined for sterol, 4α-methylsterol and triterpene alcohols. The occurrence of lupeol in high amount in the last fraction (96%) constitutes an interesting source for this compound.     

In vitro incorporation of acetate-1-14C into the phospholipids of rabbit and human endometria

Endometria from nonpregnant and 6-day pregnant rabbits and from humans in the proliferative and secretory phases were incubated with 1-¹⁴C-acetate.¹⁴CO₂ was collected, and subsequently the amounts, specific radioactivities, and in some cases the fatty acid compositions of the isolated phospholipids were determined. Phosphatidyl choline was the phospholipid present in highest amount in endometria from both nonpregnant and pregnant rabbits, and in human endometria; this phospholipid also showed the highest degree of incorporation of¹⁴C-acetate. Pregnancy in the rabbit seemed to decrease the incorporation of¹⁴C-acetate into most of the endometrial phospholipid classes. In humans, the incorporation of acetate into phosphatidyl choline and phosphatidyl ethanolamine was lower in the secretory than the proliferative endometria. Of the fatty acids, linoleic acid in phosphatidyl choline and phosphatidyl ethanolamine of the rabbit endometria showed a significant relative increase during pregnancy and palmitoleic acid showed a decrease. This investigation was supported by a grant from the Ford Foundation.     

The lipids of corn germ and endosperm

Mature kernels of an inbred corn were hand dissected into germ and endosperm fractions. Among various solvents tested, boiling, water-saturatedn-butanol extracted the most lipid from endosperm, and it was used as t h e extracting solvent for both germ and endosperm. The germ contained 78% of the total lipids and the endosperm 17%. The most striking differences in the fatty acid compositions of the triglycerides and polar lipids were higher levels of stearic and linolenic acids in the endosperm lipids. Although precautions were taken during extraction to inactivate lipases, immediately after harvest the free fatty acid level of the total lipids of the whole kernel was 6.5%. Ninety-five percent of the free fatty acids was in the endosperm fraction where the free fatty acids made up 36.5% of the total lipids. In germ, free fatty acids represented only 0.6% of the total lipids. The individual phospholipid and glycolipid classes of the endosperm and germ lipids were similar except for high levels of lyso compounds in the endosperm lipids. The higher levels of linolenic acid, free fatty acids and lyso lipids in endosperm may affect the keeping quality of the corn grain and of fractions milled from the endosperm. Presented at the AOCS meeting, St. Louis, May 1978.     

Oxysterol mediated changes in fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells

We studied the actions of oxysterols on fatty acid distribution and lipid synthesis in cultured bovine aortic smooth muscle cells. Cultures were labeled with [1-¹⁴C] arachidonate or [1-¹⁴C]oleate. During a 24-hr incubation, 25-or 22R-hydroxycholesterol enhanced the incorporation of label into triglycerides, concomitant with a reduction in the labeling of phospholipids. Cholestantriol or 20-hydroxycholesterol had the opposite effects. They caused a higher incorporation of radiolabel into phospholipids and a reduction of labeling of triacylglycerols. Similar changes were seen in cells labeled with [1-¹⁴C]acetate. Therefore, we conclude that oxysterols can promote changes in the distribution of fatty acids between neutral lipids and phospholipids through mechanisms that still need to be clarified.     

Fatty acid and cholesterol synthesis from specifically labeled leucine by isolated rat hepatocytes

Hepatocytes isolated from female rats meal-fed a high-glucose diet were incubated in Krebs-Henseleit bicarbonate medium containing 16.5 mM glucose,³H₂O, and¹⁴C-labeled amino acids (−)-Hydroxycitrate depressed the incorporation of³H₂O and [¹⁴C] alanine into fatty acids and cholesterol. Incorporation of [U-¹⁴C] leucine into lipids was not affected but incorporation of³H₂O into lipids was decreased significantly by (−)-hydroxycitrate. (−)-Hydroxycitrate depressed the incorporation of radioactivity from [2-¹⁴C]leucine into fatty acids and cholesterol by 61 and 38%, respectively, and stimulated the incorporation of radioactivity from [4,5-³H]leucine 35 and 28%. As [2-¹⁴C]leucine labels the acetyl-CoA pool and [4,5-³H]leucine labels the acetoacetate pool, it was concluded that mitochondrial 3-hydroxy-3-methylglutaryl-CoA is not incorporated intact into cholesterol, and that acetoacetate can be activated effectively in the liver cytosol for support of cholesterol and fatty acid synthesis.     

Untersuchungen über die Lipide von Kartoffeln

Investigations on the Lipids of Potatoes Gas chromatographic investigations on the fatty acid composition of the total lipids of freeze-dried potato showed that 90% of the fatty acids consist of linolenic, linoleic, palmitic and stearic acids. In all, 31 different fatty acids were detected and identified. Noticeable amounts of odd-chain fatty acids and those having more than 20 C-atoms (up to C₃₀) were found. Eight different varieties of potato were investigated. Difference in the fatty acid composition of the individual varieties was not appreciable. Experiments on the group separation of lipids showed that they contain a large amount of phospholipids (especially lecithin and cephalin). Appreciable amounts of triglycerides were also found, however, the sterol esters, sterols and free fatty acids were present to a lesser extent.     

Lipid synthesis by rat lung in vitro

Oxidation and lipogenesis in isolated rat lung tissue were studied in vitro. The minced tissue was incubated in a Krebs-Ringer bicarbonate buffer (pH 7.4) with 1-¹⁴C-acetate, 2-¹⁴C-pyruvate, U-¹⁴C-D-glucose, 1,5-¹⁴C-citrate, 1-¹⁴C-laurate, 1-¹⁴C-palmitate, 1-¹⁴C-stearate, 1-¹⁴C-oleate, 1-¹⁴C-linoleate. The lung tissue readily oxidized all of these substrates to¹⁴CO₂ and incorporated them into¹⁴C-lipids with the exception of 1,5-¹⁴C-citrate, for which there was no significant incorporation into¹⁴C-lipids. Most of the lipid¹⁴C was recovered in phospholipids, more specifically phosphatidyl choline. Twenty-eight per cent of glucose carbons was incorporated into the fatty acid moiety of phospholipids, while more than 90% of the carbons of other substrates was found in phospholipid fatty acids. The main fatty acid of the phospholipid fraction synthesized from acetate, pyruvate or glucose was palmitic acid. The oxidation of fatty acids was apparently influenced both by the carbon chain length and number of double bonds. Accumulation of¹⁴C-fatty acids in the tissue was observed when fatty acids were used as substrates; this finding suggests that the rate limiting step was not in the uptake of fatty acids. Chemical degradation of¹⁴C-myristic and palmitic acids obtained by hydrolysis of phospholipids biosynthesized from 1-¹⁴C-laurate indicated that the phospholipid fatty acids were synthesized via the de novo synthesis pathway. Presented at the AOCS-ISF World Congress, Chicago, September 1970.     

Elongation of (n−9) and (n−7)cis-monounsaturated and saturated fatty acids in seeds ofsinapis alba

Lipids in developing seeds ofSinapis alba contain appreciable proportions of (n−7)octadecenoic (vaccenic) acid besides its (n−9) isomer (oleic acid), whereas the constituent very long chain (>C₁₈) monounsaturated fatty acids of these lipids are overwhelmingly composed of the (n−9) isomers. Cotyledons of developingSinapis alba seed use [1-¹⁴C]acetate, [1-¹⁴C]malonate or [1,3-¹⁴C]malonyl-CoA for de novo synthesis of palmitic, stearic and oleic acids and for elongation of preformed oleic, vaccenic and stearic acids to their higher (n−9), (n−7) and saturated homologs, respectively. Moreover, elongation of preformed (n−7)palmitoleic acid to vaccenic acid is observed. Stepwise C₂-additions to preformed oleoyl-CoA by acetyl-CoA or malonyl-CoA yielding (n−9)icosenoyl-CoA, (n−9)docosenoyl-CoA and (n−9)tetracosenoyl-CoA are by far the most predominant reactions catalyzed by the elongase system, which seems to have a preference for oleoyl-CoA over vaccenoyl-CoA as the primer. The pattern of¹⁴C-labeling of the very long chain fatty acids formed from either acetate or malonate shows a close analogy in the mode of elongation of monounsaturated and saturated fatty acids.     

Incorporation and metabolism of fatty acids by cultured dissociated cells from rat cerebrum

Dissociated brain cells in culture incorporate a variety of saturated and unsaturated fatty acids into their cellular lipids. Of the various fatty acids studied, uptake of radioactivity was greatest for stearic acid and decreased progressively with decreasing chain length. Incorporation of radioactivity from linoliec and linolenic acids was more extensive than from oleic acid. Cellular phospholipids and triacylglycerols were labeled preferentially from all fatty acid precursors, with the relative amount of label in phospholipids being greatest when cells were incubated with linolenic acid. Fatty acids underwent desaturation and chain elongation. Changes in the labeling pattern of phospholipid fatty acids in the course of incubation demonstrated precursor-product relationships for laurate (12∶0), myristate (14∶0), palmitate (16∶0), and stearate (18∶0) and for linolenate (18∶3), eicosapentaenoate (20∶5), docosapentaenoate (22∶5), and docosahexaenoate (22∶6). The appearance of label in 22∶5 and 22∶6 paralleled the entrance of label into the ethanolamine phosphoglyceride fraction. Conversion of linoleate (18∶2 ω6) to arachidonate (20∶4 ω6) could be demonstrated but did not proceed via 18∶3 ω6.     

Linoleic acid requirement of rats fedtrans fatty acids

The amount of linoleic acid required to prevent undesirable effects of C18trans fatty acids was investigated. In a first experiment, six groups of rats were fed diets with a high content oftrans fatty acids (20% of energy [en%]), and increasing amounts of linoleic acid (0.4 to 7.1 en%). In a second experiment, four groups of rats were fed diets designed to comparetrans fatty acids with saturated andcis-monounsaturated fatty acids of the same chain length at the 2 en% linoleic acid level. After 9–14 weeks, the oxygen uptake, lipid composition and ATP synthesis of heart and liver mitochondria were determined. The phospholipid composition of the mitochondria did not change, but the fatty acid compositions of the two main mitochondrial phospholipids were influenced by the dietary fats.Trans fatty acids were incorporated in all phospholipids investigated. The linoleic acid level in the phospholipids, irrespective of the dietary content of linoleic acid, increased on incorporation oftrans fatty acids. The arachidonic acid level had decreased in most phospholipids in animals fed diets containing 2 en% linoleic acid. At higher linoleic acid intakes, the effect oftrans fatty acids on the phospholipid arachidonic acid level diminished. However, in heart mitochondrial phosphatidylethanolamine,trans fatty acids significantly increased the arachidonic acid level. Despite these changes in composition, neither the amount of dietary linoleic acid nor the addition oftrans fatty acids influenced the mitochondrial function. For rats, a level of 2 en% of linoleic acid is sufficient to prevent undesirable effects of high amounts of dietary C18trans fatty acids on the mitochondrial function.     

Differential incorporation of acetate and glucose in maturing douglas fir seeds

In order to discern the synthetic pathways of lipids in coniferous seeds, decoated maturing Douglas fir seeds were incubated with 2-¹⁴C-acetate and ul-¹⁴C-glucose for 3 hr in phosphate buffer at pH 6.0. About 52% of incorporated acetate was found in lipids, but only 9% of the absorbed glucose was converted to lipids. Distribution of incorporated radioactivity in lipid classes was similar for both substrates, 45% in polar lipids, 22% in diglycerides, 15% in triglcerides, 7% in sterol esters, 4% in each of fatty acids and monoglycerides, and 3% in sterols. High specific activity was found in free fatty acids, diglycerides, monoglycerides and polar lipids indicating a rapid turnover of the intermediates for reserve triglycerides and structural polar lipids. Degradation analyses showed that 50% of incorporated acetate and glucose in lipids were in fatty acid moiety. Acetate contributed more in sterols and other unsaponifiables than in glycerol, and the reverse was true for glucose. All the data indicated that acetate is the direct precursor of fatty acids and sterols. General synthetic pathways prevail in fir seeds. Methods for complete analysis of chemical and radio-chemical composition were presented and results discussed. Technical paper 2346 Oregon Agricultural Experiment Station.     

Incorporation of [1-14C] acetate into lipids of soybean cell suspensions

Suspension cultures of soybean cells incorporated [1-¹⁴C] acetate very rapidly into the fatty acid moieties of phospholipids and glycolipids when incubated at 26 C for up to 22 hr. The most rapidly labeled lipid was 3-sn-phosphatidylcholine, which contained 58% of the total fatty acid radioactivity after 16 min; more than 75% of this label was found to be in the oleic acid of the phosphatidylcholine. After longer periods of incubation, the proportion of¹⁴C label decreased exponentially in phosphatidylcholine and increased markedly in an unidentified phospholipid (tentatively,bis-phosphatidic acid), di- and triacylglycerols, and glycolipids. The proportion of radioactivity in oleic acid also decreased exponentially, accompanied by increases in linoleic acid first and then in linolenic acid. Most of the labeled linolenic acid at 22 hr was found in the unidentified phospholipid, di- and triacylglycerols, and the glycolipid fraction. Contribution no. 537, Ottawa Research Station, Agriculture Canada. A preliminary report was presented at the 20th International Conference on the Biochemistry of Lipids at Aberdeen, Scotland, September 1977.     

In vitro incorporation of14C-acetate into lipids of hamster flank organ (Costovertebral organ) sebaceous glands and epidermis

Following in vitro incubation of flank organs from male golden Syrian hamsters with sodium [1-¹⁴C] acetate, sebaceous glands and appendage-freed epidermis were obtained by treating the flank organ tissue with calcium chloride. This method permitted the study of incorporation of carbon-14 into the lipids of these skin components. Extracted lipids were identified by thin layer chromatography and autoradiography and were quantitated by liquid scintillation counting. Mono-, di-, and triglycerides, free sterols, fatty acids, wax monoesters, and squalene were identified as products of sebaceous gland metabolism of labeled acetate. In marked contrast, little incorporation of¹⁴C into triglycerides by the epidermal preparations was noted, although the epidermal lipids showed higher relative proportions of free sterols and polar lipids (including phospholipids). Accumulation of sterol esters did not occur. In both preparations phosphatidylcholine represented the major labeled phospholipid.